Biosensors using catalytic substances such as enzymes are being applied, not only in the analysis of glucose, but also in the analysis of various other ingredients. Such biosensors are capable of measuring various enzyme substrates precisely and easily by using minute amounts of enzymes. As opposed to traditional enzyme reactions that take place in solutions, biosensors are considered to have large economical and operational advantages as analysis systems. Among biosensors using enzymatic activity, glucose sensors are the most widely used.
Patients with diabetes, one of the adult diseases, need to measure their blood sugar level several times a day and take insulin and control the diet according to the readings. This makes glucose sensors essential for diabetic patients, as it enables easy measurement of blood glucose concentration. Glucose sensors can also be useful tools in the fields of medicine and food industry.
The glucose sensor currently available in the market was developed by Matsushita Electric Industrial Co., Ltd. This glucose sensor consists of electrodes on which an enzyme, glucose oxidase (hereafter occasionally referred to as GOD), is immobilized. In practical use, a drop of the patient's blood is placed on the sensor tip, and the blood glucose level is determined quickly after inserting the tip into a measuring device. Descriptions of this kind of glucose sensors are seen in unexamined Published Japanese Patent Application No. (JP-A) Hei 1-253648, Examined Published Japanese Patent Application No. (JP-B) Hei 5-24453, JP-A Hei 6-213858, JP-A Hei 1-156658, JP-B Hei 6-58338, JP-A Sho 63-139243, etc.
The sensor tips of these glucose sensors are fabricated by applying a solution containing glucose oxidase, potassium ferricyanide and carboxymethyl cellulose, on a silver- or carbon-paste electrode pattern printed on a board, and drying it. Potassium ferricyanide, which composes the sensor tip, is called the mediator; it is the substance that acts as an intermediary between the electrons generated through the enzymatic reaction and the electrodes. When a test solution is dropped onto the surface of the sensor tip, a reaction as in [1] below is initiated. Then, the reduced enzyme reacts with potassium ferricyanide to result in a reaction as in [2], wherein potassium ferricyanide and potassium ferrocyanide are the oxidized and reduced molecular species of each other, respectively. Lastly, the reduced mediator causes an oxidative reaction as in [3] on the electrode. Glucose level is determined by measuring this oxidation current.Glucose+Oxidated GOD->Gluconic acid+Reduced GOD  [1]Reduced GOD+Potassium ferricyanide->Oxidized GOD+Potassium ferrocyanide  [2]Potassium ferrocyanide->Potassium ferricyanide+Electron  [3]
The mediator that supports the above reactions has major roles as follows: First, it suppresses effects of other components in the test material. For example, blood contains reductive components such as ascorbic acid (vitamin C) and uric acid and they interfere with the detection of oxidation current generated by the enzymatic reaction. Use of a mediator reduces applied voltage at the time of current measurement and consequently reduces effects of interfering reductive substances. A second role of the mediator is to facilitate a sufficient enzyme reaction. Occasionally, blood of a diabetic patient shows a very high level of glucose. With a limited amount of dissolved oxygen, the high level glucose may not be sufficiently oxidized. The mediator helps enzyme-mediated glucose oxidation and thus lowers the effect of dissolved oxygen when measuring a high-level of blood glucose.
Thus, a glucose sensor using GOD is supported by the actions of the mediator. However, some problems have been pointed out concerning mediators. Low molecule weight mediators used for mediator-type biosensors are difficult to immobilize completely. Therefore, although they are claimed to be immobilized, detachment from the electrode is occasionally observed after long-term use. This makes mediator-type biosensors unsuitable for continuous use. Further, some compounds used for mediators such as ferricyanides exhibit toxicity at high concentrations. Ferrocenes, another group of compounds used for mediators, are suggested to have the danger of being poisonous due to iron deposition through the detachment. Thus, they are unsuitable for use in indwelling sensors. The risk of detachment of the immobilized substance in indwelling-type biosensors has been pointed out also regarding materials other than mediators. A detached enzyme, for example, may be recognized as a heterogenous protein and induce an allergic reaction.
In manufacturing, the use of mediators accompanies problems in uniformity of the membrane, reproducibility, adherability to the electrode, and yield ratio. That is, with the method in which the enzyme and mediator are applied onto the electrode with a carboxymethyl cellulose membrane, it is difficult to maintain a high uniformity. Further, the difficulty to maintain uniformity makes mediator-type biosensors unsuitable for compact, inexpensive sensors.
Besides biosensors using catalytic substances like enzymes those making use of reactions based on affinity between substances are also known. The former is called catalytic biosensors and the latter affinity biosensors. Representative examples of affinity biosensors are immunosensors that utilize the antigen-antibody reaction and gene sensors that utilize the affinity between nucleic acids with complementary nucleotide sequences. For immobilization of antibodies or genes in affinity biosensors, a method utilizing functional groups on the plasma-polymerized membrane applied on the sensor surface is known (Trend in Analytical Chemistry Vol. 18, pp 62–68, 1999). Functional groups accumulate at a high density on the surface of a plasma-polymerized membrane, and thus it is believed that proteins can be immobilized at a high density.
On the other hand, a catalytic biosensor with a structure of plasma-polymerized membranes overlaid on a semiconductor surface has been reported (Analytical Letters, 22, 2423–2431, 1989). In this report, although a plasma-polymerized membrane is used, the membrane is formed only on the top of the semiconductor, leaving the semiconductor pattern exposed in the cross sectional direction because a mask similar to the semiconductor pattern is used for the membrane formation. Contaminants contained in the test material affect the exposed semiconductor directly, interfering with the electrical measurement.